Lymphedema is a chronic and debilitating condition currently without approved medical therapies. Recent investigations suggest a central role for inflammation in this disease. We discovered that leukotriene B4 (LTB4), a critical lipid mediator of inflammation, promotes lymphatic endothelial cell (LEC) sprouting and growth at low concentrations (10nM), and causes LEC injury at high concentrations (200nM) by differentially affecting two essential lymphatic survival pathways, VEGFR3 and Notch. During pre-clinical lymphedema progression, lymphatic fluid LTB4 concentrations rise from initial pro-lymphangiogenic concentrations, into an anti- lymphangiogenic range which may induce pathology. Anti-LTB4 therapy reverses pre-clinical lymphedema. This finding is the scientific basis for a Phase 2 clinical trial (ULTRA), which is currently testing the efficacy of LTB4-targeted therapy for lymphedema. New results from a separate proof-of-concept clinical trial are positive, making this the first effective medicinal therapy for this condition. Even though anti-LTB4 therapy may be helpful for lymphedema, it is likely not curative, and more information is needed to understand how inflammatory pathways promote disease. Studies proposed in the grant are designed to address fundamental mechanistic questions about how LTB4 damages lymphatic capillaries and promotes a proinflammatory microenvironment. Our global hypothesis is that after lymphatic injury, increased LTB4 exacerbates lymphedema by inhibiting key lymphatic growth pathways, interfering with the (blood) microvascular circulation, transforming LECs and redirecting the immune microenvironment. To address these issues, this proposal is divided into three Specific Aims as follows. Aim 1 is to study the mechanisms by which LTB4 alters pro-lymphangiogenic signaling pathways, causes blood vascular remodeling and changes LEC cellular identity. Aim 2 will investigate how LTB4 impacts the immune microenvironment in lymphedema by influencing the activation and phenotype of proinflammatory dendritic cells and T lymphocytes and by changing lymphatic immunoregulatory functions. Finally, Aim 3 will use omic technologies to evaluate clinical lymphedema samples collected by the ULTRA trial to assess the genetic networks built around LTB4 biology in lymphedema. The goals of these studies are to understand the molecular mechanisms of reparative lymphangiogenesis, the plasticity of LEC identify and the dynamics of immune regulation in lymphedema. By carefully assessing the immune microenvironment and the global transcriptome in lymphedema, it should be possible to evolve better drug therapies for this pervasive and, otherwise, unremitting condition.